Mungbean (Vigna radiata L.), an important leguminous food crop in China, is popularly grown in arid regions. The total area of mungbean production is 8.0 × 105 ha. In August and September 2010, wilted symptoms were observed in mungbean plants in Yulin, Shaanxi Province and Datong, Shanxi Province. Infected plants had silvery gray coloration of stems and lateral branching with senesced leaves still attached to the plant. Dark brown necrotic areas were observed on the exterior of the taproot underneath the epidermis and in the pith of the lower stems of wilted plants. Black spherical microsclerotia, 43.9 μm, were present in infected plant tissues. Six fungal isolates were cultured on potato dextrose agar at 25°C and the mycelium was initially hyaline but later became gray. Black microsclerotia, 60 to 80 × 75 to 123 μm, were observed in culture after 2 to 7 days of incubation. On the basis of field symptoms, colony color, and the size of microsclerotia, the fungus was identified as Macrophomina phaseolina (Tassi) Goid (3). To confirm the morphological identification, the rDNA internal transcribed spacer (ITS) regions of the six isolates were amplified with universal primers ITS1 and ITS4. The resulting ITS sequences of the six isolates (GenBank Accession Nos. HQ660589, HQ660590, HQ660591, HQ660592, HQ660593, and HQ660594) were aligned in GenBank, which showed 97 to 99% identity with 60 M. phaseolina isolates (e.g., Accession Nos. GU046867, FJ415067, and FJ960441). Using the PCR primers MpKF1 (5′-CCGCCAGAGGACTATCAAAC-3′) and MaKR1 (5′-CGTCCGAAGCGAGGTGTATT-3′) specific for M. phaseolina (1), a 350-bp PCR fragment was obtained, indicating that these isolates were M. phaseolina. Pathogenicity tests of six isolates were performed by inoculation of 3-week-old seedlings of cv. Zhonglv 8 using the hypocotyl inoculation technique, respectively (2). Each isolate was transferred to petri dishes containing PDA 2 days prior to inoculation. On the day of inoculation, an inoculum slurry was prepared by cutting agar with the pathogen into small strips and passing the strips through a 5-syringe until uniform. A small quantity of inoculum extruded into the vertical cut in each hypocotyl of at least eight seedlings in each pot, and the PDA was used as the control to extrude into the vertical cut in each hypocotyl of at least eight seedlings in another pot. The inoculated and control plants were incubated in the mist chamber at 25°C and 90 to 100% relative humidity for 48 h before growing in a greenhouse at 30°C. Six days after inoculation, all inoculated plants, wilted or dead, showed dark brown-toblack lesions. No symptoms were observed on the control plants. For each isolate tested, M. phaseolina was reisolated from inoculated plants, but was not isolated from the control plants. The fungus has been detected in 29 plant species of 23 genera in China, including the major crops Arachis hypogaea, Helianthus annuus, and Glycine max. Although M. phaseolina has caused great yield reduction of mungbean in many areas of Asia, to our knowledge, this fungus as a causal agent of mungbean charcoal rot has not previously been reported in China.
Reference: (1) B. K. Babu et al. Mycologia 99:797, 2007. (2) D. L. Pazdernik et al. Plant Dis. 81:1112, 1997. (3) G. S. Smith and T. D.Wyllie. Charcoal rot. Page 29 in: Compendium of Soybean Diseases. 4th ed. G. L. Hartmann et al., eds. The American Phytopathological Society, St. Paul, MN, 1999.
An emerging soil-borne disease resembling Phytophthora stem rot was observed on mung bean plants grown in Anhui, China. To identify the causal agent, diseased plants and soil samples from 13 fields were collected to isolate the pathogen. Twenty-two Phytophthora isolates were recovered from the samples and conducted for detailed identification. Based on morphological and molecular characterizations, all the isolates were consistently identified as Phytophthora vignae. Phylogenetic analysis using eight nuclear loci sequences of the internal transcribed spacer (ITS) region, rRNA gene large subunit (LSU), a partial sequence of the beta-tubulin (β-tubulin) gene, the translation elongation factor 1 alpha (EF1-α), the 60S ribosomal protein L10 (60SL10), enolase (Enl) gene, the heat shock protein 90 (HSP90), and the triose phosphate isomerase/glyceraldehyde-3-phosphate dehydrogenase (TigA) and a mitochondrial locus cytochrome c oxidase subunit I (cox1) revealed that the mung bean isolates group in the same clade as P. vignae and its two formae speciales, P. vignae f. sp. adzukicola and P. vignae f. sp. vignae. A host specificity test showed that the mung bean isolates of P. vignae are pathogenic towards mung bean with a much stronger virulence and towards adzuki bean with a relatively weak virulence, but non-pathogenic to the other tested legume crops, soybean, cowpea, pea, common bean, faba bean, and chickpea. The host range of mung bean isolates significantly differs from that of the P. vignae f. sp. adzukicola and f. sp. vignae, based on our results and the previous studies. Thus, the pathogen causing Phytophthora stem rot of mung bean is proposed as a new forma specialis of P. vignae, designated as P. vignae f. sp. mungcola.
Corn (Zea mays L.) stalk rot, caused by various pathogens, is one of the most prevalent corn diseases worldwide. In October 2019, a survey was carried out to determine pathogenic fungi causing corn stalk rot in 3 fields (~120 ha) in Harbin city (44.04°N 125.42°E), Heilongjiang Province, China. In each field, 100 plants at 5 sampling points were assessed at the milk stage (R3) of development. Disease incidence was 12%. Symptomatic plants showed rapid death of the upper leaves, drooping ears and stalks were soft, hollow, watersoaked with white hyphae present on teh outside of the stalk. Pieces of tissue (0.25 cm2) from 15 individual diseased stalks (5 plants/field) were surface disinfested in 0.5% NaOCl for 5 min, rinsed three times in sterile distilled water and cultured on potato dextrose agar (PDA) containing streptomycin (50 μg/mL). After three days of incubation, a total of twelve fungal cultures with uniform characteristics were isolated and subcultured by transferring hyphal tips onto V8. Colonies on V8 selective medium were creamy white and floccus, with a growth rate of 20 mm/day at 26°C in darkness. Oospores were mostly plerotic, and oogonia walls were 1.3 to 2.7 μm thick (n = 50); globose oogonia, 23.9 to 30.5 μm in diameter (n = 50), and had 1 to 8 antheridia. Based on these characteristics, the isolates were identified as Pythium sp. (van der Plaats-Niterink 1981). Genomic DNA was extracted from single conidial cultures of representative isolates (MZYJF1, MZYJF3 and MZYJF7), and the internal transcribed spacer (ITS) region and cytochrome coxidase subunit II (CoxII) gene were amplified and sequenced using the primers ITS1/ITS4 (Yin et al. 2012) and COX2f/COX2r (Hudspeth et al. 2000), respectively. Partial nucleotide sequences of 796 bp and 573 bp for the ITS and COX11 amplicons, respectively, were obtained and deposited in GenBank (accession no. MW447501 for ITS, and MW471006 for COXII). MegaBLAST analysis of the ITS and CoxII sequences of MZYJF1 isolate showed 100% similarity with sequences from P. aristosporum strain ATCC 11101. The isolates were identified as P. aristosporum based on the fact that P. aristosporum has aplerotic oospores and less antheridia per oogonium than P. arrhenomanes (van der Plaats-Niterink 1981). A pathogenicity test was performed on corn cv. Xianyu 335 at tasseling stage (VT) in the field. An oospore suspension, obtained from isolate MZYJF1 grown on V8 agar media for 4 weeks (Green and Jensen, 2000) and diluted to 1×104 oospores/mL using blood cell counting method, was injected into the base of the maize stems of 6 healthy plants (1.5 ml/plant ) using a syringe. Control plants were injected with distilled sterile water. All inoculated plants showed symptoms 25 days after inoculation that were similar to those observed in the field. The oomycete of P. aristosporum was reisolated from symptomatic plants on V8 agar media and identified according to morphological and molecular characteristics. No symptoms were observed on the control plants. P. aristosporum has previously been reported on causing damping-off of pea in the Columbia basin of Central Washington (Alcala et al. 2016) and on soybean in North Dakota (Zitnick-Anderson and Nelson 2015). To our knowledge, this is the first report of P. aristosporum causing corn stalk rot in China. Corn stalk rot caused by P. aristosporum poses a threat to significantly reduce the quality of corn. Thus, its distribution needs to be investigated and effective disease management strategies developed.
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